Appendix 33

Appendix 33

A comparison of two 3ABC ELISA’s in an African cattle population with endemic multiple serotype Foot-and-Mouth disease

B.M.deC. Bronsvoort1, K.J. Sørensen5, J. Anderson3, A. Corteyn3, V.N. Tanya2 , R.P.Kitching4, K.L. Morgan1

1University of Liverpool, Dept. Veterinary Clinical Sciences and Animal Husbandry, Leahurst, Neston, Wirral, CH64 7TE, UK. 2Institute of Agricultural Research for Development, Regional Centre of Wakwa, B.P. 65, Ngaoundere, Cameroon. 3Institute of Animal Health, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, UK. 4National Centre for Foreign Animal Disease Canadian Food Inspection Agency Winnipeg, Manitoba, Canada. 5Danish Veterinary Institute, Virology Department, Lindholm, DK4771 Kalvehave, Denmark.

Keywords: 3ABC, CHEKIT, competitive ELISA, ROC

Abstract

The development of a foot-and-mouth disease virus (FMDV) serological test which is quick and easy to use, which can identify all 7 serotypes and which can differentiate vaccinated from convalescing or potential carriers would be a major advance in the epidemiological tool kit for FMDV. The polyprotein 3ABC has recently been proposed as such an antigen and a number of diagnostic tests are being developed. This paper compares the performance of the 3ABC FMD CHEKIT bov-ov ELISA (Bommeli) and a competitive 3ABC ELISA developed in Denmark, in an African cattle population in an endemic FMDV region of Cameroon with multiple serotypes. The test parameters (sensitivity, specificity and predictive value) were examined over a range of test cut-off’s. The results indicate that both tests lack sensitivity though the CHEKIT-ELISA is particularly low at the recommended cut-off. Their performances at different cut-off’s and how they might perform at the herd-level are discussed.

Introduction

The non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) have received considerable attention in recent years with the search for improved serological tests for FMDV 1-5. The virus neutralisation test (VNT) 6 and liquid phase blocking ELISA (LPBE) 7-9 are currently the recommended tests by the OIE. However, these tests require each serotype to be tested separately, are time consuming to perform, require virus containment facilities and cannot differentiate vaccinated from convalescing animals. The development of a single, quick to use test that covered all 7 serotypes as well as differentiating vaccinated from convalescing animals would be a major advance in the epidemiological tool kit for FMDV.

The NSP’s are only expressed in animals with replicating virus and therefore only animals that have been infected with live virus should develop antibodies to these

proteins 1,3,10,11. The currently used inactivated vaccines have been purified to remove cellular proteins and NSP’s and should not induce antibodies to these proteins. However, in practice vaccinated animals do produce antibodies to some of the NSP’s such as 3D 5 and antibodies to others such as 2C were found to rapidly fall below detectable levels 12,13. The polyproteins 3ABC and 3AB appear to be the most promising protein as diagnostic antigens 4,14-17. Recent studies of FMDV in Morocco and in Taiwan using 3ABC or 3AB ELISA’s are encouraging 18-20. If these tests are to be useful it is essential that they are evaluated in a range of populations since the diagnostic sensitivity (Se) and specificity (Sp) are population parameters that describe test performance for a given reference population 21 and previous studies have shown that when applied in tropical veterinary medicine, many diagnostic tests do not perform as well as expected 22. In addition, the overall test performance can be evaluated and the cut-off optimised for field use.

The following paper describes a comparison of the 3ABC FMD CHEKIT bov-ov ELISA1 (CHEKIT-ELISA) and a 3ABC competitive ELISA (C-ELISA) developed in Denmark. This work forms part of a larger study of the epidemiology of FMDV in the Adamawa Province of Cameroon 23. Sera from a population based sample of the cattle population of the Adamawa Province, an endemic FMDV region of Cameroon with multiple serotypes, was used to evaluate the two tests. The tests were compared to the VNT which was considered to be the ‘gold standard’. The Se (the conditional probability that an animal is test positive given it is diseased) and Sp (the conditional probability that the animal is test negative given that it is not diseased) of the two tests were estimated and the overall test performances compared using receiver operating characteristics (ROC). The test parameters are then discussed in terms of their implications when applied at the herd level.

Materials and Methods

Study Population A full description of the study area, the livestock population and the study design is given elsewhere 23. In brief, the study area was the five administrative Divisions of the 64,000km2 Adamawa Province of Cameroon. A sample frame of the cattle herds in the area was constructed from the rinderpest vaccination lists held by the local Veterinary Centres. A cross-sectional study design was used with a stratified (by Division), twostage (by Centre and Herd) random sampling strategy. The program ‘Survey Toolbox’2 , calculated a two-stage sample of 54 centres and 3 herds per centre, allowing for a 10% non-response rate. A questionnaire was administered to each herdsman and 5 juvenile (824 month old) and 5 adult (>24 months old) cattle were cast, examined for lesions and a serum and oropharyngeal fluid or probang sample (OP) taken from each animal. Five randomly selected animals gives a 95% probability of detecting at least one positive animal in a herd of 70 if the test is 100% sensitive and specific. The herds were not believed to have been vaccinated since no vaccine was available in the country and no

1 Bommeli Diagnostics, Stationsstrasse 12, CH-3097, Liebefeld, Bern, Switzerland. 2 http://www.ausvet.com.au/surveillance/toolbox.htm

herdsman reported using vaccine. The study was conducted between April and November 2000 which encompasses the rainy season when herds are close to their home areas.

Tissue culture of OP samples The OP samples were inoculated onto bovine thyroid cell monolayers following the OIE/WRL protocol. The cultures were incubated at 370C for up to 72 hours and examined for any signs of cytopathic effect (cpe). Cultures with positive cpe were then typed using the WRL sandwich antigen ELISA 24,25 .

Virus Neutralisation Test (VNT) VNT’s were performed following OIE/WRL protocol 6. The serum samples were tested against FMDV type O Manisa, type A (P59/2000-VBM/153/09) and type SAT2 (P26/2000 – FDL/74/10) respectively. The 100 TCID50 end point was estimated for each sample 26 and the standard OIE cut-off of ≥1/10-1.56 (1/45) used.

3ABC CHEKITELISA The CHEKIT-ELISA was used according to the manufacturers instructions. Briefly, the serum was diluted 1/100 and added in duplicate to the wells of a 96 well microtitre plate pre-coated with the vector expressed viral 3ABC antigen. Antibodies specific to 3ABC were bound to the antigen forming an antigen/antibody complex on the plate surface. Unbound antibody was washed away. A horseradish peroxidase labeled guinea pig antibovine IgG conjugate was added which bound to any antibody/antigen complexes. Unbound conjugate was removed by washing and the chromagen substrate added. The degree of colour that developed was proportional to the amount of antibody complexed on the plate surface and read at 405nm with a spectrophotometer. The final reading for the sample was calculated as follows using the means of the pairs of samples and the median of the 4 positive and negative controls on each plate:

value% = OD

sample − OD

neg

OD

pos − OD

neg X100

The manufacturers recommend interpretation is an %OD of less than 20% is negative, 20-30% is ambiguous and greater than 30% is positive.

FMD-3ABC Competitive ELISA The C-ELISA was performed as described previously 15 with modifications. Microtiter plates were prepared by capturing 3ABC protein produced in the Baculovirus expression system with a monoclonal antibody (MabD5) coated on the plates. Dilutions of the sera were added followed immediately by the competing antibody, horseradish peroxidase conjugated MabD5. After washing and addition of chromagen substrate (TMB H2O2) the colour development was measured and the results expressed as percentage of negative control (ODp) values.

Analysis The results of the 3 VNT’s were combined such that an animal that was positive for one or more serotypes was classed as positive on the combined VNT (cVNT). The cVNT and

ELISA results were first compared at the animal level in 2x2 tables. The Se and Sp of each test was calculated using a range of cut-offs from 10-30% for the CHEKIT-ELISA and ≤50% for the C-ELISA. The test characteristics were further investigated using the ROC calculated by using AccuROC3 .

The herd-level sensitivity (HSe) (the conditional probability that a herd is test positive given that it is diseased) and specificity (HSp) (the conditional probability that a herd is test negative given that the herd is not diseased) of the two tests were examined over a range of possible within-herd prevalences using the Herdacc4 software in a theoretical herd of 100 animals using a cut point of one positive animal.

Results

The virus isolation results from the probang samples indicated that there were at least 3 serotypes of virus actively circulating in the population and therefore VNT’s were carried out for serotypes O, A and SAT2 only. The prevalence of antibodies to these 3 serotypes are given in Table 1 below.

Table 1. Seroprevalence of serotypes O, A and SAT2 in the Adamawa Province of Cameroon. Test Juvenile Adult SAT2 42.9 (±4.7) 67.0 (±4.3) A O 12.4 (±4.4) 5.1 (±2.7) 55.8 (±7.1) 15.0 (±3.9)

The results of the C-ELISA (Table 2) and CHEKIT-ELISA (Table 3) were then compared with the cVNT results using 2x2 tables and the Se, Sp and predictive values estimated.

Table 2. The Se and Sp of the C-ELISA at 50% compared to combined VNT tests for O, A, and SAT2. 50% cut-off cVNT + cVNT- Predictive Value C-ELISA + 647 48 93.1+ % C-ELISA - 260 422 61.9- % Se=71.3% Sp=89.8%

3 AccuROC v2.4, www.accumetric.com 4 Herdacc v3.0 David Jordan, University of Guelph, Guelph, ON, Canada, N1G 2W1.

Table 3. The Se and Sp of the 3ABC at 30% (a), 20% (b), and 10% (c) compared to combined VNT tests for O, A, and SAT2 for cattle in Cameroon. (a) 30% cut-off cVNT + cVNT- Predictive Value CHEKIT + 212 10 + 95.5% CHEKIT - 695 460 - 39.8% Se=23.4% Sp=97.9%

(b) 20% cut-off cVNT + cVNT- Predictive Value CHEKIT + 314 22 + 93.5% CHEKIT - 593 448 - 43.0% Se=34.6% Sp=95.3%

(c) 10% cut-off cVNT + cVNT- Predictive Value CHEKIT + 537 74 + 87.9% CHEKIT - 370 396 - 51.7% Se=59.2% Sp=84.3%

The results show the C-ELISA to have a low sensitivity and specificity at the recommended cut-off. In comparison, the sensitivity of the CHEKIT-ELISA is extremely low at the recommended cut-off of 30% though the specificity is very high. The test cutoff’s were examined further using the ROC shown in Figure 1 below. From the curves it is clear that the C-ELISA performs better overall though the cut-off of 50% may not be optimal. The CHEKIT-ELISA though not performing as well overall is not optimised at a cut-off of 30%. Lowering the cut-off to 15% would greatly increase the sensitivity of the test without too much loss of specificity. The area under the two curves were compared using a non-parametric method 27 .

Figure 1. ROC for 3ABC CHEKIT-ELISA and C-ELISA

The estimates of the HSe and HSp of the C-ELISA and the CHEKIT-ELISA when applied to a theoretical herd of 100 animals are given in Tables 4a and 4b below. A range of herd-level prevalences were investigated from 1 to 30% for each of the tests. For the C-ELISA the low Sp is rapidly compounded with the need to increase the sample size to increase the HSe. Therefore using a cut-off of 45 or 40% would increase the Sp and overcome some of the imperfections of the test.

The CHEKIT-ELISA by having a high Sp is less prone to the rapid decline in HSp when applied to the herd. However, because the Se is also very low many more animals have to be sampled depending on the underlying herd prevalence.

Table 4. The herd-level sensitivity and specificity of the C-ELISA (a) and CHEKIT-ELISA (b) (a) Assumptions: Cut-point = 1, Sensitivity = 0.713, Specificity = 0.898. Sampling without replacement. Herd size = 100.

______________________________________________________________ Sample HSPEC HSENS: size: 0 1 3 5 10 15 30 (10) (11) (12) (13) (16) (19) (29)

______________________________________________________________ 1 0.900 0.110 0.120 0.130 0.160 0.190 0.290 11 0.294 0.742 0.774 0.802 0.869 0.914 0.982 21 0.083 0.936 0.951 0.963 0.984 0.993 1.000 31 0.020 0.987 0.992 0.995 0.999 1.000 1.000 41 0.004 0.998 0.999 0.999 1.000 1.000 1.000

(b) Assumptions: Cut-point = 1, Sensitivity = 0.234, Specificity = 0.979. Sampling without replacement. Herd size = 100.

______________________________________________________________ Sample HSPEC HSENS: size: 0 1 3 5 10 15 30 (2) (2) (3) (3) (4) (5) (8)

______________________________________________________________ 1 0.980 0.020 0.030 0.030 0.040 0.050 0.080 11 0.791 0.209 0.298 0.298 0.377 0.449 0.620 21 0.622 0.378 0.511 0.511 0.617 0.701 0.860 31 0.474 0.526 0.676 0.676 0.780 0.851 0.955 41 0.346 0.654 0.799 0.799 0.884 0.934 0.988

aColumns are HSPEC/HSENS followed by no. of infected animals in the herd. bNumbers in brackets are the predicted no. of test positives in the herd

Discussion

The results of this analysis indicate that at the individual animal level the C-ELISA performs better than the CHEKIT-ELISA in an Africa cattle population, though neither test performs particularly well. From a practical point of view there are advantages to using a competitive ELISA in that there is only one conjugate needed for all species. The additional use of a Mab as the competitor should make the standardisation of the test between laboratories much easier. As mentioned in the introduction, Se and Sp are not fixed values and will vary between sub-populations and between populations conditional on the distribution of influential covariates (eg. age, stage of disease). Therefore, it may be useful to calculate the stratum specific Se and Sp where possible or use logistic regression models that include the covariates in order to obtain estimates, as these may

have greater diagnostic utility and may be applied to other populations 21,28. It is anticipated that such analyses will be carried out.

It was not unexpected that the Se and Sp were lower than reported in previous evaluations15. This is most likely due to there being cross reactions from other pathogens/antigens which animals are exposed to in the tropics, different breeds (Bos indicus), and management systems, the multiple serotypes of FMDV that the animals were exposed to and the much wider range of disease states in a natural population21. In addition the use of the combined VNT results as a ‘gold standard’ has not been previously used and the estimates should therefore be interpreted with a degree of caution.

Although the tests are less than perfect they still may be useful depending on the circumstances in which they are used. When applying a test to a herd it is important to realise that the HSe and HSp are dependent on, the individual test Se and Sp, the number of animals tested, the true prevalence in infected herds, the herd cut-off value used to classify herds as positive and the variation in Se, Sp and prevalence among herds 29. In particular, if a test is interpreted at the herd level, as long as the individual test Sp is very high, lower individual Se’s can be overcome to some degree. So although these results can not be directly applied to the European setting the basic implications still apply.

There are clearly a number of senarios where a NSP test could be useful. For example, in endemic areas where vaccination is started as a control measure, being able to monitor the levels of sub-clinical disease would help decision making. Using the right cut-offs these tests could be used. However, as the prevalence falls, both tests but in particular the CHEKIT-ELISA will have increasing problems with low HSe unless most of the herd are sampled. Another proposed use has been at borders. The main problem with this will be that the test would need to be interpreted at the individual animal level unless all the animals were from the same source.

For Europe, which is driving much of this research, the motivation is to be able to screen vaccinated herds after an outbreak and identify herds with sub-clinical infections that could become ‘carriers’. Recent changes in OIE regulations will allow countries to resume trading after 6 months instead of 12 months if they vaccinate and can then screen and demonstrate freedom from disease. This change assumes that there is a test that will distinguish vaccinated from sub-clinically infected/convalescing animals. However, it is unclear what the prevalence of such animals would be in a herd though it is likely to be low in which case both these tests, even with adjusted cut-off’s, may struggle to achieve acceptable HSe and HSp.

APPENDIX

Apparent prevalence AP True prevalence TP Sample size per herd n

AP=(1-Sp)+(Se+Sp-1)*TP

Hse=1-(1-AP)n

HSp=Spn

Acknowledgments

The authors would like to thank Dr. Charles Nfon and Mr. Hamman Saidou Mustaffa for their considerable efforts in trying conditions in the field. In addition we thank P. Hamblin and D. Gibson for working on the VNT and ELISA. Mark Bronsvoort is a Wellcome Trust Research Training Fellow in Clinical Tropical Epidemiology.

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